Figure 4: HIF-1α is sufficient to induce lapatinib-resistance in ERBB2-expressing cells under normoxic conditions(A) Cell lysates from cells placed in hypoxia, treated with DMOG for 6 hrs, or cells expressing control or HIF-1α mutant (P402A/P564A) were collected for immunoblot analysis. (B) MCF10A-ERBB2 cells stably expressing control or HIF-1α mutant were treated with increasing doses of lapatinib and cell viability was assessed. (C) Cells as in B were stained with crystal violet. (D) Cell lysates were collected from cells as in B for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).

Mentions:
To determine whether HIF-1α stabilization is sufficient to confer lapatinib resistance in ERBB2-expressing cells under normoxic conditions, we overexpressed a stable non-degradable form of HIF-1α containing proline to alanine mutations (HIF-1α P402A, P564A) in MCF10A-ERBB2 cells. We confirmed that this mutant expressed levels similar to endogenous HIF-1α stabilized under hypoxia and to cells treated with prolyl-hydroxylase inhibitor DMOG (Figure 4A). MCF10A-ERBB2 cells expressing the stable form of HIF-1α were resistant to lapatinib-mediated effects on cell viability (Figure 4B) and cell number (Figure 4C) under normal oxygen conditions. In addition, similar to effect of hypoxia, MCF10A-ERBB2 cells expressing stable form of HIF-1α were able to maintain ERBB2, ERK, and AKT activation even when treated with high doses of lapatinib under normal oxygen conditions (Figure 4D). Stable HIF-1α expressing cells also contained reduced levels of BIM following lapatinib treatment compared to control cells (Figure 4D). Thus, HIF-1α expression alone is sufficient to block lapatinib-mediated effect on growth and signaling in ERBB2-expressing cells under normal oxygen conditions.

Figure 4: HIF-1α is sufficient to induce lapatinib-resistance in ERBB2-expressing cells under normoxic conditions(A) Cell lysates from cells placed in hypoxia, treated with DMOG for 6 hrs, or cells expressing control or HIF-1α mutant (P402A/P564A) were collected for immunoblot analysis. (B) MCF10A-ERBB2 cells stably expressing control or HIF-1α mutant were treated with increasing doses of lapatinib and cell viability was assessed. (C) Cells as in B were stained with crystal violet. (D) Cell lysates were collected from cells as in B for immunoblot analysis. Error bars indicate S.E. (*p ≤ 0.05).

Mentions:
To determine whether HIF-1α stabilization is sufficient to confer lapatinib resistance in ERBB2-expressing cells under normoxic conditions, we overexpressed a stable non-degradable form of HIF-1α containing proline to alanine mutations (HIF-1α P402A, P564A) in MCF10A-ERBB2 cells. We confirmed that this mutant expressed levels similar to endogenous HIF-1α stabilized under hypoxia and to cells treated with prolyl-hydroxylase inhibitor DMOG (Figure 4A). MCF10A-ERBB2 cells expressing the stable form of HIF-1α were resistant to lapatinib-mediated effects on cell viability (Figure 4B) and cell number (Figure 4C) under normal oxygen conditions. In addition, similar to effect of hypoxia, MCF10A-ERBB2 cells expressing stable form of HIF-1α were able to maintain ERBB2, ERK, and AKT activation even when treated with high doses of lapatinib under normal oxygen conditions (Figure 4D). Stable HIF-1α expressing cells also contained reduced levels of BIM following lapatinib treatment compared to control cells (Figure 4D). Thus, HIF-1α expression alone is sufficient to block lapatinib-mediated effect on growth and signaling in ERBB2-expressing cells under normal oxygen conditions.